Distinct genetically-determined origins of Myd88/Bcl2-driven aggressive lymphoma rationalize targeted therapeutic intervention strategies

Genomic profiling revealed the identity of at least 5 subtypes of DLBCL, including the MCD/C5 cluster characterized by aberrations in MYD88, BCL2, PRDM1 and/or SPIB. We generated mouse models harboring B cell-specific Prdm1 or Spib aberrations on the background of oncogenic Myd88 and Bcl2 lesions. We deployed whole exome sequencing, transcriptome, flow- and mass cytometry analyses to demonstrate that Prdm1- or Spib-altered lymphomas display molecular features consistent with pre-memory B cells and light zone B cells, whereas lymphomas lacking these alterations were enriched for late light-zone and plasmablast-associated gene sets. Consistent with the phenotypic evidence for increased B cell receptor signaling activity in Prdm1-altered lymphomas, we demonstrate that combined BTK/BCL2 inhibition displays therapeutic activity in mice and in five out of six relapsed/refractory DLBCL patients. Moreover, Prdm1-altered lymphomas were immunogenic upon transplantation into immuno-competent hosts, displayed an actionable PD-L1 surface expression and were sensitive to anti-murine-CD19-CAR-T cell therapy, in vivo

An autochthonous mouse model of Myd88- and BCL2-driven diffuse large B-cell lymphoma reveals actionable molecular vulnerabilities

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations

Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Since there is no common genetic alteration causing resistance to venetoclax in CLL and B cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole exome sequencing, methylated DNA immunoprecipitation sequencing and genome wide CRISPR/Cas9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter which is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity towards venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher OXPHOS and ATP production, resembling the metabolic phenotype that is seen upon venetoclax resistance. While PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity towards both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive DLBCL in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance

An Autochthonous Mouse Model of Myd88

Based on gene expression profiles, diffuse large B-cell lymphoma (DLBCL) is subdivided into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 as well as BCL2 copy-number gains. Here, we employ immune phenotyping, RNA sequencing, and wholeexome sequencing to characterize a Myd88- and BCL2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression compared with GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and PD-1 blockade significantly increased the overall survival of lymphoma-bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations. SIGNIFICANCE: Oncogenic Myd88 and BCL2 cooperate in murine DLBCL lymphomagenesis. The resulting lymphomas display morphologic and transcriptomic features reminiscent of human ABC-DLBCL. Data derived from our Myd88/BCL2-driven autochthonous model demonstrate that combined BCL2 and PD-1 blockade displays substantial preclinical antilymphoma activity, providing preclinical proof-of-concept data, which pave the way for clinical translation

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities

Based on gene expression profiles, diffuse large B-cell lymphoma (DLBCL) is subdivided into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 as well as BCL2 copy-number gains. Here, we employ immune phenotyping, RNA sequencing, and wholeexome sequencing to characterize a Myd88- and BCL2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression compared with GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and PD-1 blockade significantly increased the overall survival of lymphoma-bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations. SIGNIFICANCE: Oncogenic Myd88 and BCL2 cooperate in murine DLBCL lymphomagenesis. The resulting lymphomas display morphologic and transcriptomic features reminiscent of human ABC-DLBCL. Data derived from our Myd88/BCL2-driven autochthonous model demonstrate that combined BCL2 and PD-1 blockade displays substantial preclinical antilymphoma activity, providing preclinical proof-of-concept data, which pave the way for clinical translation

General anaesthesia versus local anaesthesia for carotid surgery (GALA): a multicentre, randomised controlled trial

Background: The effect of carotid endarterectomy in lowering the risk of stroke ipsilateral to severe atherosclerotic carotid-artery stenosis is offset by complications during or soon after surgery. We compared surgery under general anaesthesia with that under local anaesthesia because prediction and avoidance of perioperative strokes might be easier under local anaesthesia than under general anaesthesia. Methods: We undertook a parallel group, multicentre, randomised controlled trial of 3526 patients with symptomatic or asymptomatic carotid stenosis from 95 centres in 24 countries. Participants were randomly assigned to surgery under general (n=1753) or local (n=1773) anaesthesia between June, 1999 and October, 2007. The primary outcome was the proportion of patients with stroke (including retinal infarction), myocardial infarction, or death between randomisation and 30 days after surgery. Analysis was by intention to treat. The trial is registered with Current Control Trials number ISRCTN00525237. Findings: A primary outcome occurred in 84 (4·8%) patients assigned to surgery under general anaesthesia and 80 (4·5%) of those assigned to surgery under local anaesthesia; three events per 1000 treated were prevented with local anaesthesia (95% CI -11 to 17; risk ratio [RR] 0·94 [95% CI 0·70 to 1·27]). The two groups did not significantly differ for quality of life, length of hospital stay, or the primary outcome in the prespecified subgroups of age, contralateral carotid occlusion, and baseline surgical risk. Interpretation: We have not shown a definite difference in outcomes between general and local anaesthesia for carotid surgery. The anaesthetist and surgeon, in consultation with the patient, should decide which anaesthetic technique to use on an individual basis. Funding: The Health Foundation (UK) and European Society of Vascular Surgery. © 2008 Elsevier Ltd. All rights reserved

General anaesthesia versus local anaesthesia for carotid surgery (GALA): a multicentre, randomised controlled trial

BACKGROUND: The effect of carotid endarterectomy in lowering the risk of stroke ipsilateral to severe atherosclerotic carotid-artery stenosis is offset by complications during or soon after surgery. We compared surgery under general anaesthesia with that under local anaesthesia because prediction and avoidance of perioperative strokes might be easier under local anaesthesia than under general anaesthesia. METHODS: We undertook a parallel group, multicentre, randomised controlled trial of 3526 patients with symptomatic or asymptomatic carotid stenosis from 95 centres in 24 countries. Participants were randomly assigned to surgery under general (n=1753) or local (n=1773) anaesthesia between June, 1999 and October, 2007. The primary outcome was the proportion of patients with stroke (including retinal infarction), myocardial infarction, or death between randomisation and 30 days after surgery. Analysis was by intention to treat. The trial is registered with Current Control Trials number ISRCTN00525237. FINDINGS: A primary outcome occurred in 84 (4.8%) patients assigned to surgery under general anaesthesia and 80 (4.5%) of those assigned to surgery under local anaesthesia; three events per 1000 treated were prevented with local anaesthesia (95% CI -11 to 17; risk ratio [RR] 0.94 [95% CI 0.70 to 1.27]). The two groups did not significantly differ for quality of life, length of hospital stay, or the primary outcome in the prespecified subgroups of age, contralateral carotid occlusion, and baseline surgical risk. INTERPRETATION: We have not shown a definite difference in outcomes between general and local anaesthesia for carotid surgery. The anaesthetist and surgeon, in consultation with the patient, should decide which anaesthetic technique to use on an individual basis. FUNDING: The Health Foundation (UK) and European Society of Vascular Surgery